A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokine-cytokine receptor interaction signaling pathway in chronic hepatitis B

ABSTRACT Objectives: Pegylated interferon alfa (PegIFNα) has limited efficacy in patients with chronic hepatitis B (CHB). Because single-nucleotide polymorphisms (SNPs) are known to confer disease susceptibility and influence treatment response, we aimed to identify novel tripartite motif-containing 22 (TRIM22) SNPs that were associated with therapeutic efficacy in patients with CHB after PegIFNα treatment. Samples from 107 patients with CHB were genotyped using Asian Screening Array gene chips. The related mechanisms of SNPs screened were explored through both cell experiments and RNA sequence methods. TRIM22 was the most upregulated upon stimulation with PegIFNα. Specifically, the SNP rs10838543 CC genotype in TRIM22 was found to be associated with the positive response to PegIFNα treatment. The SNP rs10838543 CC genotype in TRIM22 was more stable and more robustly inhibited hepatitis B virus (HBV) replication in HepAD38 cells compared to the TT genotype. Mechanistically, we showed that the cytokine-cytokine receptor interaction signaling pathway was significantly upregulated in HepAD38 cells stably expressing the SNP rs10838543 CC genotype of TRIM22 compared to the TT genotype after PegIFNα treatment and that the SNP rs10838543 CC genotype in TRIM22 enhanced PegIFNα-induced anti-HBV activity by inducing the secretion of IFNL1, CCL3, and CCL5. The SNP rs10838543 CC genotype in TRIM22 increased the secretion of the cytokines IFNL1, CCL3, and CCL5 from hepatocytes by regulating the cytokine-cytokine receptor interaction signaling pathway and was positively correlated with the PegIFNα-induced treatment response in patients with CHB. Our findings suggest that genotyping patients with CHB for the SNP rs10838543 in TRIM22 may be a useful biomarker to help physicians identify patients who are most likely to benefit from PegIFNα treatment. IMPORTANCE Pegylated interferon alfa (PegIFNα) has limited efficacy in the treatment of chronic hepatitis B (CHB). Although many biomarkers related to hepatitis B virus (HBV) have been proposed to stratify patients, the response rate to PegIFNα is still unsatisfactory. Herein, our data suggest that the single-nucleotide polymorphism (SNP) rs10838543 in TRIM22 potentiates a positive clinical response to PegIFNα treatment in patients with hepatitis B e antigen-positive CHB by increasing the levels of IFNL1, CCL3, and CCL5. These observations can help guide treatment decisions for patients with CHB to improve the response rate to PegIFNα.

1.In Figure 1C right half of the Western blot shows stronger background staining , the author should confirmed that the six lanes with our without PegIFNα treatment were conducted parallelly without any manual modification.
2. The authors verify the transfection efficiency of their lentiviral transduction using flow cytometry (Supplemental) but provide no methods for these.
3. Part of the discussion is the repetition of the result.Please clearly pointed out the advantages and shortages of this study.
4. The English writing of the whole text needs to be polished.
Reviewer #2 (Comments for the Author): Manuscript entitled: "A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokine-cytokine receptor interaction signaling pathway in chronic hepatitis B" is well supported and interesting, the authors had experimental evidences in patients and in cell lines, and proved that rs10838543 CC genotype in TRIM22 was found to be associated with positive response to PegIFNα treatment and more robustly inhibited HBV replication in cells compared to the TT genotype.However, there are several concerns that the authors should address in current manuscript.1. Gene names should be written in italics (in lines 169 and 209, TRIM22 were formatted differently), uniform throughout, please check.2. Materials and methods should be refined, such as flow cytometry.3.As far as I know there are more than 80 members of the TRIM family, why these 14 TRIM genes were selected for the study?4. In Figure 1C, TRIM22 upregulation in the presence of IFNα in HepAD38 cells were not very visible. 5.Although 107 patient samples were used for this study, the results in the tables and figures are not representative of all 107 patients.In the results the authors state (lines 236-239), for example, that 100 people were used.The explanation needs to be given of the particular sampling done for certain analyses.6.For the RNA fold analysis, the authors showed that a nucleotide change could induce changes in the secondary structure.But the time interval of the actinomycin D test is so long that it basically reaches the plateau after 2 hours, and it is recommended to shorten the time interval.

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Thank you for submitting your paper to Microbiology Spectrum.This research entitled "A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokine-cytokine receptor interaction signaling pathway in chronic hepatitis B" aimed to find and explain the potential marker for differential PegIFNα response.The study is generally well designed, and demonstrated that the rs10838543 CC genotype in TRIM22 increased secretion of the cytokines IFNL1, CCL3, and CCL5 from hepatocytes by regulating the cytokine-cytokine receptor interaction signaling pathway and was positively correlated with the PegIFNα-induced treatment response in patients with CHB.The study might be interesting for readers since it suggest that genotyping patients with CHB at rs10838543 polymorphism might be a potential biomarker helping physicians identify patients who are most likely to benefit from PegIFNα treatment.But there are still some concerns regarding this study: 1.In Figure 1C right half of the Western blot shows stronger background staining , the author should confirmed that the six lanes with our without PegIFNα treatment were conducted parallelly without any manual modification.
2. The authors verify the transfection efficiency of their lentiviral transduction using flow cytometry (Supplemental) but provide no methods for these.
3. Part of the discussion is the repetition of the result.Please clearly pointed out the advantages and shortages of this study.
4. The English writing of the whole text needs to be polished.
Manuscript entitled: "A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokine-cytokine receptor interaction signaling pathway in chronic hepatitis B" is well supported and interesting, the authors had experimental evidences in patients and in cell lines, and proved that rs10838543 CC genotype in TRIM22 was found to be associated with positive response to PegIFNα treatment and more robustly inhibited HBV replication in cells compared to the TT genotype.
However, there are several concerns that the authors should address in current manuscript.
1. Gene names should be written in italics (in lines 169 and 209, TRIM22 were formatted differently), uniform throughout, please check.
2. Materials and methods should be refined, such as flow cytometry.
3. As far as I know there are more than 80 members of the TRIM family, why these 14 TRIM genes were selected for the study?4. In Figure 1C, TRIM22 upregulation in the presence of IFNα in HepAD38 cells were not very visible.
5. Although 107 patient samples were used for this study, the results in the tables and figures are not representative of all 107 patients.In the results the authors state (lines 236-239), for example, that 100 people were used.The explanation needs to be given of the particular sampling done for certain analyses.
6.For the RNA fold analysis, the authors showed that a nucleotide change could induce changes in the secondary structure.But the time interval of the actinomycin D test is so long that it basically reaches the plateau after 2 hours, and it is recommended to shorten the time interval.Response: Thank you for your constructive suggestion.In the revised manuscript, we have added the following text to discuss the advantages and shortages of this study: In this study, we demonstrated that a novel TRIM22 gene polymorphism, SNP rs10838543 in TRIM22, promotes the response to PegIFNα therapy by elevating levels of IFNL1, CCL3, and CCL5 through cytokine-cytokine receptor interaction signaling pathway in CHB patients.Genotyping patients with CHB for the SNP rs10838543 in TRIM22 may be a useful biomarker to help physicians identify patients who are more likely to response to PegIFNα therapy.(Discussion, pages 15, lines

312-317)
There are still some limitations to our current study that will be addressed in further research.First, TRIM22 is expressed only in human and nonhuman primates, and, owing to a lack of a license for breeding tree shrews, we were unable to validate our findings in vivo.Future studies should prioritize controlled in vivo studies to confirm the relevance of the SNP rs10838543 genotypes in TRIM22 for PegIFNα therapy.
Second, further delineation of the molecular mechanism that results in differential IFNL expression in the context of different SNP rs10838543 genotypes in TRIM22, and whether this might occur due to inhibition of suppressors of cytokine signaling (SOCS) 1, SOCS2, or SOCS3, is warranted.(Discussion, page 18, lines 377-378, line

382)
4. The English writing of the whole text needs to be polished.

Response:
We apologize for the language problems in the original manuscript.In the revised manuscript, we have improved the language presentation with the assistance from a native English speaker with appropriate research background.Response: Thank you for your important comment.In the revised manuscript, we have supplemented the methods for flow cytometry in the Supplemental material as follow:

Transfection efficiency of the lentiviral transduction
Evaluation of transduction efficiency was conducted using green fluorescent protein (GFP) expressed in all lentiviruses.Cells were washed three times with PBS, and then dissociated with trypsin.After cell suspensions by DMEM, cells were transferred to suitable FACS tubes.A negative control was performed using untransfected HepAD38 cells.FITC fluorescence was analyzed by flow cytometry and expressed as a percentage.(Supplemental Material, methods, page 3, lines 45-50) 3.As far as I know there are more than 80 members of the TRIM family, why these 14 TRIM genes were selected for the study?
Response: This is an important question.There were indeed more than 80 members of the TRIM families (DOI: 10.1016/j.tibs.2017.01.002).However, the aim of this study was to find genes and their polymorphisms associated with PegIFNα efficacy.
In our previous work, we screened only 14 TRIM genes with differences between the CR and SR groups by ASA microarray.Therefore, we selected these 14 TRIM genes for the study.Response: We apologize for the confusion.Due to the limitations of ethics and sample size, each specimen was unable to be tested simultaneously for both Asian Screening Array (ASA) gene chips and validation of various candidate gene biomarkers.In this study, we performed peripheral blood samples using ASA gene chips.Then, we verified the levels of these differentially expressed genes by qRT-PCR.Our study was limited by the variable volumes of patient sample available, and with each additional test and validation performed, remaining sample volume further decreased past usable levels, preventing us from uniformly assaying all patient samples.
6.For the RNA fold analysis, the authors showed that a nucleotide change could induce changes in the secondary structure.But the time interval of the actinomycin D test is so long that it basically reaches the plateau after 2 hours, and it is recommended to shorten the time interval.
Response: Thanks for your very thoughtful suggestion.In the revised manuscript, according to the reviewer's comment, cells were seeded in 6-well plates at a density of 2×10 5 cells per well for 24 hours, then cells were collected at 0 h, 0.5 h, 1 h, 1.5 h, 2 h and 3 h after actinomycin D treatment.The mRNA level of TRIM22 was detected by qRT-PCR.We found that the half-life of mRNA of SNP rs10838543 CC genotype in TRIM22 (t 1/2 =1.50 h) was still significantly longer than TT genotype (t 1/2 =0.57h) (Fig. 2F Re: Spectrum02247-23R1 (A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokinecytokine receptor interaction signaling pathway in chronic hepatitis B) Dear Mr. Qishui Ou: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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Reviewer # 2 :
Manuscript entitled: "A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokine-cytokine receptor interaction signaling pathway in chronic hepatitis B" is well supported and interesting, the authors had experimental evidences in patients and in cell lines, and proved that rs10838543 CC genotype in TRIM22 was found to be associated with positive response to PegIFNα treatment and more robustly inhibited HBV replication in cells compared to the TT genotype.However, there are several concerns that the authors should address in current manuscript.1. Gene names should be written in italics (in lines 169 and 209, TRIM22 were formatted differently), uniform throughout, please check.Response: Thank you for your suggestion.In the revised manuscript, we have scrutinized the full text and made the appropriate formatting changes.(Line 3and methods should be refined, such as flow cytometry.

Fig. 1 .
Fig. 1.PegIFNα treatment upregulates TRIM22 expression Fig. 2. The SNP rs10838543 CC genotype in TRIM22 has higher TRIM22 mRNA • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred